Dipeptide derivatives

ABSTRACT

Compounds of the formula  
                 
 
wherein R, R 1 , COOR 2 , R 3 -R 7 , alk, and X have meaning as defined, such being useful as dual inhibitors of angiotensin converting enzyme and neutral endopeptidase, as well as inhibitors of endothelin converting enzyme.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of provisional application No.60/291,088 filed May 15, 2001 and of provisional application No.60/339,575 filed Dec. 11, 2001, the contents of which are incorporatedherein by reference.

SUMMARY OF THE INVENTION

The present invention directed to novel vasopeptidase inhibitorsdescribed below which are useful as dual inhibitors of both angiotensinconverting enzyme (ACE) and neutral endopeptidase (NEP, EC 3.4.24.11).The compounds of the invention are particularly useful for the treatmentand/or the prevention of conditions which are responsive to ACE and NEPinhibition, particularly cardiovascular disorders, such as hypertension,isolated systolic hypertension, renal failure (including edema and saltretention), pulmonary edema, left ventricular hypertrophy, heart failure(including congestive heart failure) and atherosclerosis.

The compounds of the invention are also useful for reducing elevatedcholesterol plasma levels in mammals. Furthermore, they also inhibitendothelin converting enzyme (ECE) and are useful for the treatmentand/or prevention of conditions which are responsive to ECE inhibition.

By virtue of their inhibition of neutral endopeptidase, the compounds ofthe invention may also be useful for the treatment of pain, depression,certain psychotic conditions, and cognitive disorders. Other potentialindications include the treatment of angina, premenstrual syndrome,Meniere's disease, hyperaldosteronism, hypercalciuria, ascites,glaucoma, asthma and gastrointestinal disorders such as diarrhea,irritable bowel syndrome and gastric hyperacidity.

By virtue of their inhibition of ECE, the compounds of the invention mayalso be useful for the treatment and/or prevention of endothelindependent conditions and diseases, including cerebral ischemia (stroke),subarachnoid hemorrhage, traumatic brain injury, cerebral vasospasm,arterial hypertrophy, restenosis, Raynaud's disease, myocardialinfarction, obesity; also prostate hyperplasia, migraine, diabetesmellitus (diabetic nephropathy), preeclampsia, glaucoma, andtransplantaUon rejection such as in aorta or solid organtransplantation; as well as erectile dysfunction.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compounds of formula I

-   -   wherein    -   R represents hydrogen, lower alkyl, carbocyclic or heterocyclic        aryl-lower alkyl or cycloalkyl-lower alkyl;    -   R₁ represents lower alkyl, cycloalkyl, carbocyclic or        heterocyclic aryl, or biaryl; or R₁ represents (cycloalkyl,        carbocyclic aryl, heterocyclic aryl or biaryl)-lower alkyl;    -   alk represents lower alkylene;    -   R₃ represents hydrogen or acyl;    -   R₄ represents hydrogen, optionally substituted lower alkyl,        carbocyclic or heterocyclic aryl, (carbocyclic or heterocyclic        aryl)-lower alkyl, cycloalkyl, cycloalkyl-lower alkyl, biaryl,        biaryl-lower alkyl, oxacycloalkyl, thiacycloalkyl,        azacycloalkyl, or (oxacycloalkyl, thiacyctoalkyl or        azacycloalkyl)-lower alkyl;    -   R₅ represents hydrogen or lower alkyl; or    -   R₄ and R₅ together with the carbon atom to which they are        attached, represent cycloalkylidene, benzo-fused        cycloalkylidene; or 5- or 6-membered (oxacycloalkylidene,        thiacycloalkylidene or azacycloalkylidene), each optionally        substituted by lower alkyl or aryl-lower alkyl;    -   R₆ represents lower alkyl, carbocyclic or heterocyclic aryl,        (carbocyclic or heterocyclic aryl)-lower alkyl, cycloalkyl,        cycloalkyl-lower alkyl, biaryl or biaryl-lower alkyl;    -   R₇ represents lower alkyl, (carbocyclic or heterocyclic        aryl)-lower alkyl, cycloalkyl-lower alkyl or biaryl-lower alkyl;        or    -   R₆ and R₇, together with the carbon atom to which they are        attached, represent 3- to 10-membered cycloalkylidene which may        be substituted by lower alkyl or aryl-lower alkyl or may be        fused to a saturated or unsaturated carbocyclic 5- to 7-membered        ring; or 5- or 6-membered (oxacycloalkylidene,        thiacycloalkylidene or azacycloalkylidene), each optionally        substituted by lower alkyl or aryl-lower alkyl; or        2,2-norbonylidene;    -   X represents —O—, —S(O)_(n)—, —NHSO₂—, or —NHCO—;    -   n is zero, one or two; and    -   COOR₂ represents carboxyl or carboxyl derivatized in form of a        pharmaceutically acceptable ester;    -   disulfide derivatives derived from said compounds wherein R₃ is        hydrogen; and pharmaceutically acceptable salts thereof.

The present invention is also directed to pharmaceutical compositionscomprising said compounds; methods for preparation of said compounds;intermediates; and methods of treating disorders in mammals which areresponsive to ACE and NEP inhibition by administration of said compoundsto mammals in need of such treatment.

Encompassed by the instant invention are also any prodrug derivatives ofcompounds of the invention having a free carboxyl, sulfhydryl or hydroxygroup, said prodrug derivatives being convertible by solvolysis or underphysiological conditions to be the free carboxyl, sulfhydryl and/orhydroxy compounds. Prodrug derivatives are, e.g., the esters of freecarboxylic acids and S-acyl and O-acyl derivatives of thiols, alcoholsor phenols, wherein acyl has meaning as defined herein.

Pharmaceutically acceptable esters are preferably prodrug esterderivatives, such being convertible by solvolysis or under physiologicalconditions to the free carboxylic acids of formula I.

Pharmaceutically acceptable prodrug esters are preferably, e.g., loweralkyl esters, aryl-lower alkyl esters, α-(lower alkanoyloxy)-lower alkylesters such as the pivaloyloxymethyl ester, and α-(lower alkoxycarbonyl,morpholinocarbonyl, piperidinocarbonyl, pyrrolidinocarbonyl or di-loweralkylaminocarbonyl)-lower alkyl esters.

Pharmaceutically acceptable salts are salts derived frompharmaceutically acceptable bases for any acidic compounds of theinvention, e.g., those wherein COOR₂ represents carboxyl. Such are,e.g., alkali metal salts (e.g., sodium, potassium salts), alkaline earthmetal salts (e.g., magnesium, calcium salts) amine salts (e.g.,tromeihamine salts).

Compounds of formula I, depending on the nature of substituents, possesstwo or more asymmetric carbon atoms. The resulting diastereomers andoptical antipodes are encompassed by the instant invention. Thepreferred configuration is indicated in formula Ia.

wherein asymmetric carbons carrying the substituents—alk-X—R, and R₄typically have the S-configuration.

Preferred are the compounds of formula I and Ia wherein R and RSrepresent hydrogen; R₁ represents lower alkyl, C₅- or C₆-cycloalkyl,carbocyclic or heterocyclic aryl, or (carbocyclic or heterocyclicaryl)-lower alkyl; alk represents lower alkylene; X represents —O— or—S(O)_(n) wherein n represents zero or two; R₃ represents hydrogen oracyl; R₄ represents optionally substituted lower alkyl, oxacycloalkyl,oxacycloalkyl-lower alkyl, or (carbocyclic or heterocyclic aryl)-lweralkyl; R₅ represents hydrogen, or R₄ and R₅ combined with the carbonatom to which they are attached represent C₅ or C₆-cycloalkylidene; R₆and R₇ represent lower alkyl; or R₆ and R₇, together with the carbonatom to which they are attached, represent 5- or 6-memberedcycloalkylidene; COOR₂ represents carboxyl or carboxyl derivatized inform of a pharmaceutically acceptable ester; disulfide derivativesderived from said compounds wherein R₃ is hydrogen; and pharmaceuticallyacceptable salts thereof.

Further preferred are said compounds of formula I and Ia wherein R andR₅ represent hydrogen; R₁ represents carbocyclic or heterocyclic aryl or(carbocyclic or heterocyclic aryl)-lower alkyl; R₃ represents hydrogenor optionally substituted lower alkanoyl; R₄ represents lower alkyl,cycloalkyl, tetrahydropyranyl or C₁-C₄-lower alkoxy-lower alkyl; R₆ andR₇ both represent C₁-C₄-alkyl and are identical; X represents —O— or—S—; alk represents methylene; COOR₂ represents carboxyl, loweralkoxycarbonyl, (di-lower alkylaminocarbonyl)-lower alkoxycarbonyl or(morpholinbcarbonyl, piperidinocarbonyl or pyrrolidinocarbonyl)-loweralkoxycarbonyl; and pharmaceutically acceptable salts thereof.

Particularly preferred are said compounds of formula I or Ia wherein Rand R₅ represent hydrogen; R₁ represents carbocyclic aryl or carbocyclicaryl-lower alkyl in which carbocyclic aryl represents phenyl or phenylsubstituted by one or two of hydroxy, lower alkanoyloxy, lower alkyl,lower alkoxy, trifluoromethyl, trifluoromethoxy or halo; R₃ representshydrogen, lower alkanoyl or lower alkanoyl substituted by lower alkoxy;R₄ represents lower alkyl, 4-tetrahydropyranyl or C₁-C₄-loweralkoxy-C₁-C₄-lower alkyl; R₆ and R₇ represent methyl; X represents —O—;alk represents methylene or ethylene; and COOR₂ represents carboxyl orlower alkoxycarbonyl; and pharmaceutically acceptable salts thereof. Anembodiment thereof relates to compounds wherein R₃ represents hydrogenor lower alkanoyl.

Further preferred are the above compounds of formula I or Ia wherein Rand R₅ represent hydrogen; R₁ represents phenyl fluorophenyl, benzyl orfluorobenzyl; R₃ represents hydrogen, lower alkanoyl or lower alkanoylsubstituted by lower alkoxy; R₄ represents isopropyl, tert-butyl,1-methoxyethyl or 4-tetrahydropyranyl; R₆ and R₇ represent methyl; Xrepresents —O—; alk represents methylene; and COOR₂ represents carboxylor lower alkoxycarbonyl; and pharmaceutically acceptable salts thereof.An embodiment thereof relates to compounds wherein R₃ representshydrogen or lower alkanoyl.

Preferred particular embodiments relate to compounds of formula I or Iawherein R and R₅ represent hydrogen; R₁ represents benzyl; R₃ representshydrogen, acetyl or methoxyacetyl; R₄ represents isopropyl ortert-butyl; R₆ and R₇ represent methyl; X represents —O—; alk representsmethylene; and COOR₂ represents carboxyl or ethoxycarbonyl; or apharmaceutically acceptable salt thereof.

The definitions as such or in combination as used herein, unless denotedotherwise, have the following meanings within the scope of the presentinvention.

Aryl represents carbocyclic or heterocyclic aryl, either monocyclic orbicyclic.

Monocyclic carbocyclic aryl represents optionally substituted phenyl,being preferably phenyl or phenyl substituted by one to threesubstituents, such being advantageously lower alkyl, hydroxy, loweralkoxy, acyloxy, halogen, cyano, trifluoromethyl, trifluoromethoxy,amino, lower alkanoylamino, lower alkyl-(thio, sulfinyl or sulfonyl),lower alkoxycarbonyl, mono- or di-lower alkylcarbamoyl, or mono- ordi-lower alkylamino; or phenyl substituted by lower alkylenedioxy.

Bicyclic carbocyclic-aryl represents 1- or 2-naphthyl or 1- or2-naphthyl preferably substituted by lower alkyl, lower alkoxy orhalogen.

Monocyclic heterocyclic aryl represents preferably optionallysubstituted thiazolyl, pyrimidyl, triazolyl, thienyl, furanyl orpyridyl.

Optionally substituted furanyl represents 2- or 3-furanyl or 2- or3-furanyl preferably substituted by lower alkyl.

Optionally substituted pyridyl represents 2-, 3- or 4-pyridyl or 2-, 3-or 4-pyridyl preferably substituted by lower alkyl, halogen or cyano.

Optionally substituted thienyl represents 2- or 3-thienyl or 2- or3-thienyl preferably substituted by lower alkyl.

Optionally substituted pyrimidyl represents, e.g., 0.2-pyrimidyl or2-pyrimidyl substituted by lower alkyl.

Optionally substituted thiazolyl represents, e.g., −2-thiazolyl or2-thiazolyl substituted by lower alkyl.

Optionally substituted triazolyl represents, e.g., 1,2,4-triazolyl or1,2,4-triazolyl preferably substituted by lower alkyl.

Bicyclic heterocyclic aryl represents preferably indolyl,benzothiazolyl, quinolinyl or isoquinolinyl optionally substituted byhydroxy, lower alkyl, lower alkoxy or halogen, advantageously 3-indolyl,2-benzothiazolyl or 2- or 4-quinolinyl.

Aryl as in aryl-lower alkyl is preferably phenyl or phenyl substitutedby one or two of lower alkyl, lower alkoxy, hydroxy, lower alkanoyloxy,halogen, trifluoromethyl, cyano, lower alkanoylamino or loweralkoxycarbonyl; also, optionally substituted naphthyl.

Aryl-lower alkyl is advantageously benzyl or 1- or 2-phenethyloptionally substituted on phenyl by one or two of lower alkyl, loweralkoxy, hydroxy, lower alkanoyloxy, halogen or trifluoromethyl.

The term “lower” referred to herein in connection with organic radicalsor compounds respectively defines such with up to and including 7,preferably up and including 4 and advantageously one or two carbonatoms. Such may be straight chain or branched.

Optionally substituted lower alkyl refers to lower alkyl or lower alkylsubstituted by, e.g., halo, hydroxy, lower alkoxy, amino, (mono- ordi-lower alkyl) amino, acylamino, 1-lower alkyl-piperazino, morpholino,piperidino, pyrrolidino and the like.

Lower alkylene refers to a straight or branched carbon chain havingpreferably 1 to 4 carbon atoms, which may be substituted, e.g., by loweralkoxy, for example, —CH₂—, —CH (CH₃)—, —CH₂CH₂— and the like.

A lower alkyl group preferably contains 1-4 carbon atoms which may bestraight chain or branched and represents, for example, ethyl, propyl,butyl or advantageously methyl.

A lower alkoxy group preferably contains 1-4 carbon atoms which may bestraight chain or branched and represents, for example, methoxy,propoxy, isopropoxy or advantageously ethoxy.

Cycloalkyl represents a saturated cyclic hydrocarbon radical whichpreferably contains 5- to 7-ring carbons, preferably cyclopentyl orcyclohexyl.

Oxacycloalkyl represents preferably 5- to 7-membered oxacycloalkyl,e.g., tetrahydropyranyl, such as 4-tetrahydropyranyl.

Thiacycloalkyl represents preferably 5- to 7-membered thiacycloalkyl,e.g., tetrahydrothiopyranyl, such as 4-tetrahydrothiopyranyl.

Azacycloalkyl represents preferably 5- to 7-membered azacycloalkyl,e.g., pyrrolidinyl or piperidinyl in which the nitrogen may besubstituted by lower alkyl or aryl-lower alkyl.

The term cycloalkyl-lower alkyl represents preferably (cyclopentyl orcyclohexyl)-methyl, 1- or 2-(cyclopentyl or cyclohexyl)ethyl, 1-, 2- or3-(cyclopentyl or cyclohexyl)propyl, or 1-, 2-, 3- or 4-(cyclopentyl orcyclohexyl)-butyl. Similarly (oxacyclyl, thiacycloalkyl orazacycloalkyl)-lower alkyl.

A lower alkoxycarbonyl group preferably contains 1 to 4 carbon atoms inthe alkoxy portion and represents, for example, methoxycarbonyl,propoxycarbonyl, isopropoxycarbonyl or advantageously ethoxycarbonyl.

Cycloalkylidene is 3- to 10-membered, preferably 5- or 6-membered, andrepresents a cycloalkane linking group in which the two attached groupsare attached to the same carbon of the cycloalkane ring.

5- or 6-membered oxacycloalkylidene represents a tetrahydrofuran ortetrahydropyran linking group, i.e., tetrahydrofuranylidene ortetrahydropyranylidehe, in which the two attached groups are attached tothe same carbon atom of the respective rings, e.g., at the 3- or4-position thereof.

5- or 6-membered thiacycloalkylidene represents a tetrahydrothiophene ortetrahydrothiopyran linking group in which the two attached groups areattached to the same carbon atom of the respective rings, e.g., at the3- or 4-position thereof.

5- or 6-membered azacycloalkylidene represents a pyrrolidine orpiperidine linking group in which the two attached groups are attachedto the same carbon atom of the respective rings, e.g., at the 3- or4-position thereof, and the nitrogen may be substituted by lower alkyl,e.g., methyl, or by aryl-lower alkyl, e.g., benzyl.

Benzo-fused cycloalkylidene represents, e.g., 1,1- or2,2-tetralinylidene or 1,1- or 2,2-indanylidene.

Halogen (halo) preferably represents fluoro or chloro, but may also bebromo or iodo.

Acyl is derived from a carboxylic acid and represents preferablyoptionally substituted lower alkanoyl, carbocyclic aryl-lower alkanoyl,aroyl, lower alkoxycarbonyl or aryl-lower alkoxycarbonyl, advantageouslyoptionally substituted lower alkanoyl, or aroyl.

Lower alkanoyl is preferably acetyl, propionyl, butyryl, or pivaloyl.

Optionally substituted lower alkanoyl, for example, represents loweralkanoyl or lower alkanoyl substituted by, e.g., lower alkoxycarbonyl,lower alkanoyloxy, lower alkanoylthio, lower alkoxy, lower alkylthio,hydroxy, di-lower alkylamino, lower alkanoylamino, morpholino,piperidino, pyrrolidiho, 1-lower alkylpiperazino, aryl or heteroaryl.

Aroyl is carbocyclic or heterocyclic aroyl, preferably monocycliccarbocyclic or monocyclic heterocyclic aroyl.

Monocyclic carbocyclic aroyl is preferably benzoyl or benzoylsubstituted by lower alkyl, lower alkoxy, halogen or trifluoromethyl.

Monocyclic heterocyclic aroyl is preferably pyridylcarbonyl orthienylcarbonyl.

Acyloxy is preferably optionally substituted lower alkanoyloxy, loweralkoxycarbonyloxy, monocyclic carbocyclic aroyloxy or monocyclicheterocyclic aroyloxy.

Aryl-lower alkoxycarbonyl is preferably monocyclic carbocyclic-loweralkoxycarbonyl, advantageously benzyloxycarbonyl.

Biaryl represents monocarbocyclic aryl substituted by monocycliccarbocyclic or monocyclic heterocyclic aryl, and preferably representsbiphenylyl, advantageous 4-biphenylyl optionally substituted on one orboth benzene rings by lower alkyl, lower alkoxy, halogen ortrifluoromethyl.

Biaryl-lower alkyl is preferably 4-biphenylyl-lower alkyl,advantageously 4-biphenylyl-methyl.

The novel compounds of the invention are ACE inhibitors inhibiting theconversion of angiotensin I to the pressor substance angiotensin II andthus decrease blood pressure in mammals. Furthermore, compounds of theinvention demonstrate inhibition of NEP and thus potentiate thecardiovascular (e.g., diuretic and natriuretic) effects of atrialnatriuretic factors (ANF). The combined effect is beneficial for thetreatment of cardiovascular disorders in mammals, in particular,hypertension, cardiac conditions such as congestive heart failure, andrenal failure. A further beneficial effect of the compounds of theinvention in the treatment of said cardiovascular disorders is theinhibition of ECE.

The above-cited properties are demonstrable in vitro and in vivo tests,using advantageously mammals, e.g., mice, rats, dogs, monkeys, orisolated organs, tissues and preparations thereof. Said compounds can beapplied in vitro in the form of solutions, e.g., preferably aqueoussolutions, and in vivo either enterally, parenterally, advantageously,orally (p.o.) or intravenously (i.v.), e.g., as a suspension or inaqueous solution. The dosage in vitro may range between about 10⁻⁶ molarand 10⁻⁹ molar concentrations. The dosage in vivo may range, dependingon the route of administration, between about 0.01 and 50 mg/kg,advantageously between about 0.1 and 25 mg/kg.

In vitro testing is most appropriate for the free carboxylic acids ofthe invention. The test compound is dissolved in dimethyl sulfoxide,ethanol or 0.25 M sodium bicarbonate solution, and the solution isdiluted with buffer to the desired concentration.

The in vitro inhibition of the AGE by the compounds of this inventioncan be demonstrated by a method analogous to that given in Biochem.Pharmacol., Vol. 20, p. 1637 (1971). The buffer for the ACE assay is 300mM NaCl, 100 mM KH₂PO₄ (pH 8.3). The reaction is initiated by theaddition of 100 μL of hippuryl-histidyl-leucine (2 mg/mL) to tubescontaining enzyme and drug in a volume of 150 μL and tubes are incubatedfor 30 minutes at 37° C. The reaction is terminated by the addition of0.75 mL 0.6 N NaOH. 100 μL of freshly prepared O-pthaldehyde solution (2mg/mL in methanol) is added to the tubes, the contents are mixed andallowed to stand at room temperature. After 10 minutes, 100 μL of 6 NHCl is added. The tubes are centrifuged and the supernatant opticaldensity is read at 360 nM. The results are plotted against drugconcentration to determine the IC₅₀, i.e., the drug concentration whichgives half the activity of the control sample containing no drug.

Typically, the compounds of invention demonstrate an IC₅₀ in the rangeof about 0.1-50 nM for ACE inhibition.

Illustrative of the invention, the compound of Example 6(a) demonstratesan IC₅₀ of about 20 nM in the ACE in vitro assay.

Inhibition of ACE can be demonstrated in vivo on p.o. or i.v.administration by measuring inhibition of the angiotensin I inducedpressor response in normotensive rats.

The in vivo test for i.v. administered compounds is performed with Male,normotensive rats, which are conscious. A femoral artery and femoralvein are cannulated respectively for direct blood pressure measurementon i.v. administration of angiotensin I and i.v. or p.o. admiriistrationof a compound of this invention. After the basal blood pressure isstabilized, pressor responses to 3 or 4 challenges of 300 ng/kgangiotensin I i.v., at 15-minute intervals, are obtained. Such pressureresponses are usually again obtained at 15, 30, 60 and 90 minutes, andthen every hour up to 6 hours after i.v. or p.o. administration of thecompound to be tested, and compared with the initial responses. Anyobserved decrease of said pressor response is an indication of ACEinhibition.

Illustrative of the invention, the compound of Example 6(a) inhibits theangiotensin I induced pressor response for 3 hours at a dose of 10 mg/kgi.v. Similarly, the compound of Example 1 (a) inhibits the angiotensin Iinduced pressor response for 6 hours at a dose of 11.8 mg/kg p.o.

The in vitro inhibition of NEP (EC 3.4.24.11) can be determined asfollows:

NEP 3.4.24.11 activity is determined by the hydrolysis of the substrateglutaryl-Ala-Ala-Phe-2-naphthylamide (GAAP) using a modified procedureof Orlowski and Wilk (1981). The incubation mixture (total volume 125μL) contains 4.2 μL of protein (rat kidney cortex membranes prepared bymethod of Maeda et al., 1983), 50 mM tris buffer, pH 7.4 at 25° C., 500μM substrate (final concentration), and leucine aminopeptidase M (2.5μg). The mixture is incubated for 10 minutes at 25° C., and 100 μL offast garnet (250 μg fast garnet/mL of 10% Tween 20 in 1 M sodium acetatepH 4.2) is added. Enzyme activity is measure spectrophotometrically at540 nM. One unit of NEP 24.11 activity is defined as 1 nmol of2-naphthylamine released per minute at 25° C. at pH 7.4. IC₅₀ values aredetermined, i.e., the concentration of test compound required for 50%inhibition of the release of 2-naphthylamine.

NEP activity can also be determined using ANF as a substrate. ANFdegrading activity is determined by measuring the disappearance ofrat-ANF (r-ANF) using a 3-minute reverse phase-HPLC separation. Analiquot of the enzyme in 50 mM tris HCl buffer, pH 7.4, is pre-incubatedat 37° C. for 2 minutes and the reaction is initiated by the addition of4 nmol of r-ANF in a total volume of 50 μL. The reaction is terminatedafter 4 minutes with the addition of 30 μL of 0.27% trifluoroacetic acid(TFA). One unit of activity is defined as the hydrolysis of 1 nmol ofr-ANF per minute at 37° C. at pH 7.4. IC₅₀ values are determined, i.e.,the concentration of test compound required for 50% inhibition of thehydrolysis of ANF.

Typically, the compounds of the invention demonstrate an IC₅₀ in therange of about 0.1-50 nM for NEP inhibition.

Illustrative of the invention, the compound of Example 6(a) demonstratesan IC₅₀ of about 5 nM in the GMP in vitro assay.

The effect of the compounds of the invention on rat plasma ANFconcentration can be determined as follows:

Male Sprague-Dawley rats (275-390 g) are anesthetized with ketamine (150mg/kg)/acepromazine (10%) and instrumented with catheters in the femoralartery and vein to obtain blood samples and infuse ANF, respectively.The rats are tethered with a swivel system and are allowed to recoverfor 24 hours before being studied in the conscious, unrestrained state.

In the assay, plasma ANF levels are determined in the presence andabsence of NEP inhibition. On the day of study, all rats are infusedcontinuously with ANF at 450 ng/kg/min. i.v. for the entire 5 hours ofthe experiment. Sixty minutes after beginning the infusion, bloodsamples for baseline ANF measurements are obtained (time 0) and the ratsare then randomly divided into groups treated with the test compound orvehicle. Additional blood samples are taken 30, 60,120,180 and 240minutes after administration of the test compound.

Plasma ANF concentrations are determined by a specific radioimmunoassay.The plasma is diluted (×12.5, ×25 and ×50) in buffer containing: 50 mMtris (pH 6.8), 154 mM NaCl, 0.3% bovine serum albumin, 0.01% EDTA. Onehundred microliters of standards [rANF (99-126)] or samples are added to100 μL of rabbit anti-rANF serum and incubated at 4° C. for 16 hours.Ten thousand cpm of [¹²⁵I]rANF are then added to the reaction mixturewhich is incubated at 4° C. for 16 hours. Ten thousand cpm of [¹²⁵I]rANFare then added to the reaction mixture which is incubated at 4° C. foran additional 24 hours. Goat anti-rabbit IgG serum coupled toparamagnetic particles is added to the reaction mixture and bound[¹²⁵I]rANF is pelleted by exposing the mixture to an attracting magneticrack. The supernatant is decanted and the pellets counted in a gammacounter. All determinations are performed in duplicate. Plasma ANFlevels are expressed as a percent of those measured in vehicle-treatedanimals which received ANF alone (450 ng/kg/min. i.v.)

Illustrative of the invention, the compound of Example 1 (a) increasesplasma ANF levels by about 70% at a dose of 11.8 mg/kg p.o.

The anti-hypertensive activity can be determined, e.g., in thespontaneously hypertensive rat (SHR) and the DOCA-salt hypertensive rat,e.g., according to Bazil et al., J. Cardiovasc. Pharmacol., Vol. 22, pp.897-905 (1993) and Trapani et al., J. Cardiovasc. Pharmacol., Vol. 14,pp. 419-424 (1989), respectively.

Illustrative of the invention, the compound of example 1(a) reduces meanarterial pressure in conscious SHR at once daily administration of 11.8mg/kg p.o.

The anti-hypertensive effect can be determined in desoxy-corticosteroneacetate (DOCA)-salt hypertensive rats as follows:

DOCA-salt hypertensive rats (280-380 g) are prepared by the standardmethod. Rats undergo a unilateral nephrectomy and one week later areimplanted with silastic pellets containing 100 mg/kg of DOCA. The ratsare maintained on 1% of NaCl/0.2% KCl drinking water for three to fiveweeks until sustained hypertension is established. The anti-hypertensiveactivity is evaluated at this time.

Two days before an experiment, the rats are anesthetized withmethoxyflurane and instrumented with catheters in the femoral artery tomeasure arterial blood pressure. Forty-eight hours later, baselinearterial pressure and heart rate are recorded during a one hour period.The test compound or vehicle is then administered and the samecardiovascular parameters are monitored for an additional 5 hours.

The diuretic (saluretic) activity can be determined in standard diureticscreens, e.g., as described in “New Anti-hypertensive Drugs”, SpectrumPublications, pp. 307-321 (1976), or by measuring the potentiation ofANF-induced natriuresis and diuresis in the rat.

The potentiation of the natriuretic effect of ANF can determined asfollows:

Male Sprague-Dawley rats (280-360 g) are anesthetized with inactin (100mg/kg i.p.) and instrumented with catheters in the femoral artery,femoral vein and urinary bladder to measure arterial pressure,administer ANF and collect urine, respectively. A continuous infusion ofnormal saline (33 μL/min.) is maintained throughout the experiment topromote diuresis and sodium excretion. The experimental protocolconsists of an initial 15-minute collection period (designated aspre-control) followed by three additional collection periods.Immediately after completion of the pre-control period, test compound orvehicle is administered; nothing is done for the next 45 minutes. Then,blood pressure and renal measurements are obtained during a secondcollection period (designated control, 15 minutes). At the conclusion ofthis period, ANF is administered (1 μg/kg i.v. bolus) to all animals andarterial pressure and renal parameters are determined during twoconsecutive 15-minute collection periods. Mean arterial pressure, urineflow and urinary sodium excretion are determined for all collectionperiods. Blood pressure is measured with a Gould p50 pressuretransducer, urine flow is determined gravimetrically, sodiumconcentration is measured by flame photometry, and urinary sodiumexcretion is calculated as the product of urine flow and urine sodiumconcentration.

The in vitro inhibition of ECE can be determined as follows:

ECE is partially purified from porcine primary aortic endothelial cellsby DE52 anion exchange column chromatography and its activity isquantified by radioimmunoassay (RIA) as described in Anal. Biochem.,Vol., 212, pp. 434-436(1993). Alternatively, the native enzyme can besubstituted by a recombinant form of ECE, as described, for example, inCell, Vol. 78, pp. 473-485 (1994). Human ECE-1 has been described byseveral groups (Schmidt et al., FEBS Letters, Vol. 356, pp. 238-243(1994); Kaw et al., 4th Int. Conf. on Endothelin; April 23-25, London(UK) (1995) C6; Valdenaire et al., J. Biol. Chem., Vol. 270, pp.29794-29798 (1995); Shimada et al., Biochem. Biophys. Res. Commun., Vol.207, pp. 807-812 (1995)). The ECE inhibition can be determined asdescribed in Biochem. Mol. Biol. Int., Vol. 31, No. 5, pp. 861-867(1993), by RIA to measure ET-1 formed from big ET-1.

Alternatively, recombinant human ECE-1 (rhECE-1) can be used, asfollows:

Chinese hamster ovary cells expressing rhECE-1 (Kaw et al., 4th Int.Conf. on Endothelin; April 23-25, London (UK), (1995) C6) are culturedin DMEM/F12 medium containing 10% fetal bovine serum and 1×antibiotic-antimycotic. Cells are harvested by scraping, pelleted bycentrifugation, and homogenized at 4° C. in a buffer containing 5 mMMgCl₂, 1 μM pepstatin A, 100 μM leupeptin, 1 mM PMSF, and 20 mM Tris, pH7.0, with a ratio of 2 mL of buffer/mL of cells. The cell debris isremoved by brief centrifugation, and the supernatant is centrifugedagain at 100,000×g for 30 minutes. The resulting pellet is re-suspendedin a buffer containing 200 mM NaCl and 50 mM Tes, pH 7.0, at a proteinconcentration about 15 mg/mL and stored in aliquots at −80° C.

To assess the effect of an inhibitor on ECE-1 activity, 10 μg of proteinis pre-incubated with the compound at a desired concentration for 20minutes at room temperature in 50 mM TES, pH 7.0, and 0.005% TritonX-100 in a volume of 10 μL. Human big ET-1 (5 μL) is then added to afinal concentration of 0.2 μM, and the reaction mixture is furtherincubated for 2 hours at 37° C. The reaction is stopped by adding 500 μLof RIA buffer containing 0.1% Triton X-100, 0.2% bovine serum albumin,and 0.02% NaN₃ in phosphate-buffered saline.

Diluted samples (200 μL) obtained from the above enzyme assay areincubated at 4° C. overnight with 25 μL each of [¹²⁵I]ET-1 (10,000cpm/tube) and 1:20,000-fold diluted rabbit antibodies that recognizespecifically the carboxyl terminal tryptophan of ET-1. Goat anti-rabbitantibodies coupled to magnetic beads (70 μg) are then added to eachtube, and the reaction mixture is further incubated for 30 minutes atroom temperature. The beads are pelleted using a magnetic rack. Thesupernatant is decanted, and the radioactivity in the pellet is countedin a gamma counter. Total and nonspecific binding are measured in theabsence of non-radioactive ET-1 and anti-ET antibodies, respectively.Under these conditions, ET-1 and big ET-1 displace [¹²⁵I]ET-1 binding tothe antibodies with IC₅₀ values of 21±2 and 260,000±66,000 fmol(mean±SEM, n=3-5), respectively.

In order to determine the IC₅₀ value of an inhibitor, aconcentration-response curve of each inhibitor is determined. AnIBM-compatible version of ALLFIT program is used to fit data to aone-site model.

ECE inhibition can also be determined in vivo by measuring theinhibition of big ET-1-induced pressor response in the anesthesized orconscious rat, as described below. The effect of the inhibitors on thepressor response resulting from big ET-1 challenge is measured inSprague-Dawley rats as described in Biochem. Mol. Biol. Int., Vol. 31,No. 5, pp. 861-867 (1993). Results are expressed as percent inhibitionof the big ET-1-induced pressor response as compared to vehicle.

Male Sprague-Dawley rats are anesthetized with Inactin (100 mg/kg i.p.)and instrumented with catheters in the femoral artery and vein to recordmean arterial pressure (MAP) and administer compounds, respectively. Atracheostomy is performed and a cannula inserted into the trachea toensure airway patency. The body temperature of the animals is maintainedat 37±1° C. by means of a heating blanket. Following surgery, MAP isallowed to stabilize before interrupting autonomic neurotransmissionwith chlorisondamine (3 mg/kg i.v.). Rats are then treated with the testcompound at 10 mg/kg i.v. or vehicle and challenged with big ET-1 (1nmol/kg i.v.) 15 and 90 minutes later. Generally, the data are reportedas the maximum increase in MAP produced by big ET-1 in animals treatedwith the test compound or vehicle.

Male Sprague-Dawley rats are anesthetized with methohexital sodium (75mg/kg i.p.) and instrumented with catheters in the femoral artery andvein to measure MAP and administer drugs, respectively. The cathetersare threaded through a swivel system that enables the rats to movefreely after regaining consciousness. The rats are allowed to recoverfrom this procedure for 24 hours before initiating the study. On thefollowing day, MAP is recorded via the femoral artery catheter and atest compound or vehicle is administered via the femoral vein. Animalsare challenged with big ET-1 at 1 nmolkg i.v. at various times afterdosing. After an adequate washout period, depending upon the dose andregimen, animals can be re-tested at another dose of test compound orvehicle. Generally, the data are reported as the change in MAP producedby big ET-1 at 2-minute intervals in animals treated with the testcompound as compared to vehicle.

ECE inhibition can also be determined in vivo by measuring theinhibition of the big ET-1 induced pressor response in conscious SHR,e.g. as described in Biochem. Biophys. Res. Commun., Vol. 204, pp.407-412 (1994).

Male SHR (16-18 weeks of age) are administered either test compound orvehicle (1 M NaHCO₃) via an osmotic minipump implanted subcutaneously.On day 5, femoral arterial and venous catheters are placed inanesthetized rats for the measurement of MAP and for test compoundadministration, respectively. After a 48-hour recovery period, MAP isrecorded (day 7) through the arterial catheter connected to a pressuretransducer. Blood pressure and heart rate are allowed to stabilize for30 minutes before ganglion blockade is performed using chlorisondamine(10 mg/kg i.v.). Approximately 15 minutes later, a bolus dose of bigET-1 (0.25 nmol/kg i.v.) is administered to both vehicle- and testcompound-treated rats. The change in blood pressure in response to bigET-1 is then compared between the two groups of rats.

The inhibition of cerebral vasospasm is demonstrated by measuring theinhibition of experimentally induced constriction of basilar cerebralarteries in the rabbit (Caner et al., J. Neurosurg., Vol. 85,pp.-917-922 (1996).

The degree or lack of undesirable immunostimulatory potential of thecompounds of the invention can be determined with the murine popliieallymph node assay described in Toxicology Letters, Vols. 112/113, pp.453-459 (2000).

The compounds of the invention, e.g., can be prepared

-   -   a) by condensing a compound of formula II        wherein the symbols alk, X, R, R₁, R₆ and R₇ have the meaning as        defined above and COOR₂ represents esterified carboxyl, with a        carboxylic acid of the formula III        or a reactive functional derivative thereof, wherein R₄ and R₅        have meaning as defined above; R₃′ represents hydrogen or a        labile S-protecting group, e.g., acyl, t-butyl or optionally        substituted benzyl; or    -   b) by condensing a compound of the formula IV        or a reactive functional derivative thereof wherein the symbols        R₃′, R₄-R₅ and R₆-R₇ have meaning as defined above, with an        amino acid ester of the formula V        wherein alk, X, R and R₁ have meaning as defined above and COOR₂        represents esterified carboxyl; or    -   c) by condensing under basic conditions a compound of the        formula VI        wherein the symbols R, R₁, COOR₂, R₄-R₇, alk and X have meaning        as defined above and Y represents a reactive esterified hydroxyl        group (e.g., chloro or bromo) as a leaving group, with a        compound of the formula        R₃′SH  (VII)        or a salt thereof, wherein R₃ represents a labile S-protecting        group, e.g., acyl, t-butyl or optionally substituted benzyl; and        converting a resulting product to a compound of formula I        wherein R₃ is hydrogen;        and in above said process, if temporarily protecting any        interfering reactive group(s), removing said protecting        group(s), and then isolating the resulting compound of the        invention; and, if desired, converting any resulting compound of        the invention into another compound of the invention; and/or, if        desired, converting a free carboxylic acid function into a        pharmaceutically acceptable ester derivative, or converting a        resulting ester into the free acid or into another ester        derivative; and/or, if desired, converting a resulting free        compound into a salt or a resulting salt into the free compound        or into another salt, and/or, if desired, separating a mixture        of isomers or racemates, and/or, if desired, resolving a        racemate obtained into the optical antipodes.

In starting compounds and intermediates which are convened to thecompounds of the invention in manner described herein, functional grouppresent, such as thiol, carboxyl, amino and hydroxy groups, areoptionally protected by conventional protecting groups that are commonin preparative organic chemistry. Protected thiol, carboxyl, amino andhydroxy groups are those that can be converted under mild conditionsinto free thiol, carboxyl, amino and hydroxy groups without otherundesired side reactions taking place.

The purpose of introducing protecting groups is to protect thefunctional groups from undesired reactions with reaction components andunder the conditions used for carrying out a desired chemicaltransformation. The need and choice of protecting groups for aparticular reaction is known to those skilled in the art and depends onthe nature of the functional group to be protected (thiol, carboxyl,amino group, etc.), the structure and stability of the molecule of whichthe substituent is a part, and the reaction conditions.

Well-known protecting groups that meet these conditions and theirintroduction and removal are described, for example, in J. F. W. McOmie,“Protective Groups in Organic Chemistry”, Plenum Press, London, N.Y.(1973), T. W. Greene and P. G. M. Wuts, “Protective Groups in OrganicSynthesis”, Wiley, N.Y. 3^(rd) Ed. (1999), and also in “The Peptides”,Vol. I, Schroeder and Luebke, Academic Press, London, N.Y. (1965).

The preparation of compounds of the invention according to process (a)involving the condensation of an amine of formula II with the acid offormula III or a functional reactive derivative thereof, is carried outby methodology well-known for peptide synthesis.

The condensation according to process (a) of an amino ester of formulaII with a free carboxylic acid of formula III is carried outadvantageously in the presence of a condensing agent such asdicyclohexylcarbodiimide,N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide, hydroxybenzotriazole,1-hydroxy-7-azabenzotriazole, chlorodimethoxytriazine,benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(BOP Reagent), orO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU), either alone or in combination, andtriethylamine or N-methylmorpholine, in an inert polar solvent, such asethyl acetate, acetonitrile, dimethylformamide or methylene chloride,preferably at room temperature.

The condensation of an amino ester of formula II with a reactivefunctional derivative of an acid of formula III in the form of an acidhalide, advantageously an acid chloride, or mixed anhydride, is carriedout in an inert solvent such as toluene or methylene chloride,advantageously in the presence of a base, e.g., an inorganic base suchas potassium carbonate or an organic base such as triethylamine,N-methylmorpholine or pyridine, preferably at room temperature.

Reactive functional derivatives of carboxylic acids of formula III arepreferably acid halides (e.g., the acid chloride) and mixed anhydrides,such as the pivaloyl or isobutyloxycarbonyl anhydride, or activatedesters such as benzotriazole, 7-azabenzotriazole or hexafluorophenylester.

The starting material of formula II can be prepared according to methodsdescribed herein and illustrated in the examples.

The preparation of a starting material of formula II involves theacylatidn of an ester of formula VIII

wherein alk, X, R and R₁ have meaning as defined hereinabove and COOR₂represents esterified carboxyl (e.g., wherein R₂ is lower alkyl orbenzyl) with an appropriately N-protected amino acid (or a reactivefunctional derivative) of formula IX

wherein R₆ and R₇ have meaning as defined hereinabove and R₈ is a labileamino protecting group, e.g., t-butoxycarbonyl, to obtain thecorresponding N-protected compound of formula II.

The condensation of a compound of formula VIII with a compound offormula IX is carried out by methodology well-known in peptidesynthesis, e.g., as described above for the condensation of a compoundof formula II with a compound of formula III. The N-protecting group isremoved according to methods well-known in the art, e.g., thet-butoxycarbonyl is removed with anhydrous acid such as trifluoroaceticacid or HCl.

The starting amino esters and acids of compounds of formula VIII and IX,respectively, are either known in the art, or if new, can be preparedaccording to methods well-known in the art, e.g., or illustrated herein.The amino acid esters of formula VIII are preferably the S-enantiomers.

The starting materials of formula III are known, or if new, may beprepared according to conventional methods. The starting materials areprepared, e.g., from the corresponding racemic or optically activeα-amino acids, by conversion thereof to the α-bromo derivative followedby displacement thereof with inversion of configuration using theappropriate thiol derivative of formula VII, under basic conditions, forexample, as illustrated in European Patent Application No. 524,553published Jan. 27, 1993. S-debenzylation of the resulting final productsis carried out by reductive cleavage, e.g., with Raney nickel inethanol. S-deacylation is carried out by, e.g., base catalyzedhydrolysis with dilute aqueous sodium hydroxide. Cyclic startingmaterials of formula III can be prepared by treatment of the cycliccarboxylic acid (e.g., cyclopentanecarboxylic acid) with sulfur in thepresence of a strong base such as lithium diethylamide.

The preparation of the compounds of the invention according to process(b) involving the condensation of an acid of formula IV with an aminoacid ester of formula V is carried out in a similar fashion to process(a). Similarly, the starting materials of formula IV are prepared bycondensation of an acid of formula III with an ester corresponding togem-disubstituted amino acids of formula IX (wherein R₈ is now hydrogen)under conditions similar to those described above, followed by removalof the carboxylprotecting group.

The preparation of the compounds of the invention according to process(c) involving the displacement of a leaving group Y in a compound offormula VI with a thiol derivative R₃′-SH as a salt thereof is carriedout according to methods well-known in the art.

A reactive esterified hydroxyl group, represented by Y, is a hydroxylgroup esterified by a strong inorganic or organic acid. Corresponding Ygroups are in particular halo, for example, chloro, bromo or iodo, alsosulfonyloxy groups, such as lower alkyl- or arylsulfonyloxy groups, forexample, (methane-, ethane-, benzene- or toluene-) sulfonyloxy groups,also the trifluoromethylsulfonyloxy group.

The displacement is carried out in an inert solvent, such asdimethylformamide or methylene chloride in the presence of a base suchas potassium carbonate, triethylamine, diisopropylethylamine,N-methylmorpholine, and the like at room or elevated temperature. Usinga salt of R₃′SH (e.g., potassium thioacetate), the reaction is carriedout in the absence of a base, in an inert solvent such astetrahydrofuran or dimethylformamide.

Similarly, the starting materials of formula VI can be prepared byreacting the dipeptide derivative of formula II with an acid of theformula

wherein R₁₄ and R₅ and Y have meaning as defined above, under conditionsdescribed for process (a).

The compounds of formula X wherein Y is halo, such as theα-bromocarboxylic acids are known and are prepared, e.g., as describedin International Application WO 99/55726 published Nov. 4, 1999.

The compounds of the invention and intermediates, e.g., those offormulas II, V and VI, having the side chain alk-X—R₁ are prepared fromthe corresponding compounds having the alk-X′ side chain wherein X′represents amino, hydroxy, thiol or a suitable leaving group accordingto methodology known in the art and illustrated herein. For example, theacids and esters of formula V can be obtained starting with serine,homoserine, threonine, cysteine and the like, preferably in opticallyactive form.

Certain compounds of the invention and intermediates can be converted toeach other according to general reactions well-known in the art.

The free mercaptans may be converted to the S-acyl derivatives byreaction with a reactive derivative of a carboxylic acid (correspondingto R₃ being acyl in formula I), such as an acid anhydride or saidchloride, preferably in the presence of a base such as triethylamine inan inert solvent such as acetonitrile or methylene chloride.

Free alcohols and phenols can be converted to the corresponding acylderivatives, e.g., by reaction with a corresponding acid chloride in thepresence of a base, such as triethylamine.

The free mercaptans, wherein R₃ represents hydrogen, may be oxidized tothe corresponding disulfides, e.g., by air oxidation or with the use ofmild oxidizing agents such as iodine in alcoholic solution. Conversely,disulfides may be reduced to the corresponding mercaptans, e.g., withreducing agents such as sodium borohydride, zinc and acetic acid ortributylphosphine.

Carboxylic acid esters may be prepared from a carboxylic acid bycondensation with, e.g., the halide corresponding to R₂—OH, in thepresence of a base, or with an excess of the alcohol in the presence ofan acid catalyst, according to methods well-known in the art.

Carboxylic acid esters and S-acyl derivatives may be hydrolyzed, e.g.,with aqueous alkali such as alkali metal carbonates or hydroxides.S-acyl and ester groups can be selectively removed as illustratedherein.

Preferably, and wherever possible, the preferred isomers of theinvention of formula Ia are prepared from pure enantiomers.

In case mixtures of stereoisomers (e.g., diastereomers) are obtained,these can be separated by known procedures such as fractionalcrystallization and chromatography (e.g., thin layer, column, flashchromatography). Racemic free acids can be resolved into the opticalantipodes by fractional crystallization of d- or I-(α-methylbenzylamine,cinchonidine, cinchonine, quinine, quinidine, dehydroabiethylamine,brucine or strychnine) salts and the like. Racemic products, if notdiastereoisomers, can first be converted to diastereoisomers withoptically active reagents (such as optically active alcohols to formesters) which can then be separated as described above, and, e.g.,hydrolyzed to the individual enantiomer. Racemic products can also beresolved by chiral chromatography, e.g., high pressure liquidchromatography using a chiral absorbent; also by enzymatic resolution,e.g., of esters with alkalase.

The above-mentioned reactions are carried out according to standardmethods, in the presence or absence of diluents, preferably such as areinert to the reagents and are solvents thereof, of catalysts, alkalineor acidic condensing or said other agents respectively and/or inert tothe reagents and are solvents thereof, of catalysts, alkaline or acidiccondensing or said other agents respectively and/or inert atmospheres,at low temperatures, room temperature or elevated temperatures,preferably near the boiling point of the solvents used, at atmosphericor superatmospheric pressure.

The invention further includes any variant of said processes, in whichan intermediate product obtainable at any stage of the process is usedas a starting material and any remaining steps are carried out, or theprocess is discontinued at any stage thereof, or in which the startingmaterials are formed under the reaction conditions, or in which thereaction components are used in the form of their salts or opticallypure antipodes. Mainly those starting materials should be used in saidreactions, that lead to the formation of those compounds indicated aboveas being preferred.

The present invention additionally relates to the use in mammals of thecompounds of the invention and their pharmaceutically acceptable,non-toxic acid addition salts, or pharmaceutical compositions thereof,as medicaments, for inhibiting both ACE and NEP, and, e.g., for theprevention or treatment of cardiovascular disorders such ashypertension, edema, salt retention and congestive heart failure, eitheralone or in combination with one or more other agents which are usefulfor the treatment of such disorders. Such may be anti-hypertensiveagents, anti-atherosclerotic agents, cardiac agents, diuretic agents,antidiabetic agents, cholesterol-lowering agents and the like. When usedin combination with other therapeutic agents such can be administeredseparately or in a fixed combination.

Examples of therapeutic agents which can be used in combination areangiotensin II receptor antagonists, such as valsartan, losartan,candesartan, eprosartan, irbesartan and telmisartan; β-blockers, such asbisoprolol, propanolol, atenolol, sotalol and metoprolol; renininhibitors; calcium channel blockers, such as amlodipine, verapamil,diltiazem, bepridil, felodipine, isradipine, nicardipine, nifedipine,nimodipine and nisoldipine; aldosterone synthase inhibitors/aldosteroneantagonists, such as eplerenone, (+)-fadrozole (WO 01/76574),spironolactone and canrenone; diuretics, such as furosemide,hydrochlorothiazide, indapamide, metazolone, amiloride and triamterene;vasopressin receptor antagonists, such as OPC 21268, SR 49059,SR121463A, SR49059, VPA985, OPC31260 and YM087; cardiotonic drugs, suchas enoximone and levosimendan; endothelin antagonists and ECEinhibitors, such as bosentan, BMS193884, TBC3711 and compounds disclosedin WO 99/55726; anti-atherosclerotic agents, particularly cholesterollowering agents, such as bile acid sequestrants (e.g., cholestyramineand colestipol); cholesterol absorption inhibitors, such as ezetimibe;fibrates, such as fehofibrate and gemfibrozil; statin HMG CoA reductaseinhibitors, such as atorvastatin, fluvastatin, lovastatin, pravastatin,simvastatin and pitavastatin; and nicotinic acid derivatives;thyromimetic agents, such as those disclosed in U.S. Pat. No. 5,569,674and WO 00/58279; also antidiabetic agents, such as repaglinide,nateglinide, metformin, rosiglitazone, pioglitazone, glyburide,glipizide, glimepiride, DPP728, LAF237, NH622 and DRF4158.

The present invention also relates to the use of the compounds of theinvention for the preparation of pharmaceutical compositions, especiallypharmaceutical compositions having ACE and NEP inhibiting activity, and,e.g., anti-hypertensive activity.

The pharmaceutical compositions according the invention are thosesuitable for enteral, such as oral or rectal, transdermal and parenteraladministration to mammals, including man, for the treatment ofcardiovascular disorders, such as hypertension, comprising an effectiveamount of a pharmacologically active compound of the invention or apharmaceutically acceptable salt thereof, alone or in combination withone or more pharmaceutically acceptable carriers, as well as incombination with other therapeutic agents also useful for the treatmentof cardiovascular disorders, as indicated above.

The pharmacologically active compounds of the invention are useful inthe manufacture of pharmaceutical compositions comprising an effectiveamount thereof in conjunction or admixture with excipients or carrierssuitable for either enteral or parenteral application. Preferred aretablets and gelatin capsules comprising the active ingredient, togetherwith a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol,cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearicacid, its magnesium or calcium salts and/or polyethyleneglycol; fortablets also c) binders, e.g., magnesium aluminum silicate, starchpaste, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose and/or polyvinylpyrrolidone; if desired, d)disintegrants, e.g., starches, agar, alginic acid or its sodium salt, oreffervescent mixtures; and, if desired, absorbents, colorants, flavorsand sweeteners. Injectable compositions are preferably aqueous isotonicsolutions or suspensions, and suppositories are advantageously preparedfrom fatty emulsions or suspensions. Said compositions may be sterilizedand/or contain adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure and/or buffers. In addition, the compositions may also containother therapeutically valuable substances. Said compositions areprepared according to conventional mixing, granulating or coatingmethods, respectively, and contain about 0.1-75%, preferably about1-50%, of the active ingredient.

Suitable formulations for transdermal application include an effectiveamount of a compound of the invention with carrier. Advantageouscarriers include absorbable pharmacologically acceptable solvents toassist passage through the skin of the host. Characteristically,transdermal devices are in the form of a bandage comprising a backingmember, a reservoir containing the compound, optionally with carriers,optionally a rate controlling barrier to deliver the compound to theskin of the host at a controlled and predetermined rate over a prolongedperiod of time, and means to secure the device to the skin.

A unit dosage for a mammal of about 50-70 kg may contain between about10 and 200 mg of the active ingredient. The dosage of active compound isdependent on the species of warm-blooded animal (mammal), the bodyweight, age and individual condition, and on the form of administration.

The following examples are intended to illustrate the invention and arenot to be construed as being limitations thereon. Temperatures are givenin degrees Centigrade. If not mentioned otherwise, all evaporations areperformed under reduced pressure, preferably between about 15 and 100 mmHg. Optical rotations (expressed in degrees) are measured at roomtemperature at 589 nM (D line of sodium) or other wave lengths asspecified in the examples. The structure of the compounds are confirmedby standard analytical methods such as mass spectrum, elementalanalysis, NMR, IR spectroscopy and the like.

The prefixes R and S are used to indicate the absolute configuration ateach asymmetric center.

EXAMPLE 1 (a)N-[2-[(S)-2-Acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-O-benzyl-L-serineethyl ester

N-[2-[-[(R)-3-bromo-3-methylbutanoylamino]-2-methylproprionyl-O-benzyl-L-serineethyl ester (4.96 g, 10.5 mmol) is dissolved in tetrahydrofuran (100 mL)and potassium thioacetate (6.00 g, 52.5 mmol) is added. The mixture isstirred at room temperature for 4 hours, then diluted with ethyl acetate(500 mL) and washed with water (100 mL), sodium bicarbonate solution(2×100 mL), water (2×100 mL) and then brine (50 mL). The solution isdried over sodium sulfate and concentrated in vacuo. The crude materialis purified by flash chromatography (silica gel, 3:2 hexane/ethylacetate) to yield title compound; m.p. 55-570C; [α]²⁰ _(D)−63.5°(c=0.99, CH₃OH); MS (M+H): 467.

Alternately, the above displacement can be carried out with 2equivalents of potassium thioacetate in ethyl acetate at roomtemperature and the resulting product can be crystallized from t-butylmethyl ether/heptane (40/60) to give the title compound having m.p. of68° C.

The starting material is prepared as follows:

A solution of O-benzyl-L-serine (9.75 g, 50 mmol) in ethanol (200 mL) issaturated with HCl gas for 8 minutes. The mixture is stirred overnightat room temperature, and then concentrated in vacuo. The solid is washedwith diethyl ether and collected by filtration to yieldO-benzyl-L-serine ethyl ester hydrochloride as a white solid.

To a solution of BOC-α-methylalanine (3.05 g, 15 mmol),O-benzyl-L-serine ethyl ester hydrochloride (3.89 g, 15 mmol),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCl, 2.88 g, 15 mmol)and 1-hydroxy-7-azabenzotriazole (HOAT, 2.04,15 mmol) in methylenechloride (150 mL) is added triethylamine (1.52 g, 15 mmol). The mixtureis stirred overnight and then concentrated in vacua. The residue isre-dissolved in ethyl acetate and washed with water, 1 N HCl, water, andbrine. The solution is dried over sodium sulfate and concentrated toyield N-[2-(BOC-amino)-2-methylpropionyl]-O-benzyl-L-serine ethyl esterof the formula

Alternately, the above carbamate can be prepared by condensingO-benzyl-L-serine ethyl ester hydrochloride with BOC-α-methylalanine inthe presence of 1 equivalent of 2-chloro-4,6-dimethoxy-1,3,5-triazine(CDMT, see Synthesis, pp. 917-920 (1987)) and N-methylmorpholine (2.5equivalents) in ethyl acetate at room temperature.

The above carbamate (6.12 g, 15 mmol) is dissolved in methylene chloride(200 mL) and chilled in an ice bath. The solution is saturated with HClgas for 10 minutes and then stirred at room temperature overnight. Theresidue is concentrated. Methylene chloride is added and the residue isconcentrated again to giveN-(2-amino-2-methylpropionyl)-O-benzyl-L-serine ethyl esterhydrochloride as a foam; MS (M+H): 309.

Alternately, N-(2-amino-2-methylpropionyl)-O-benzyl-L-serinehydrochloride can be prepared by treating the carbamate with HCl gas (3equiv.) in ethyl acetate at a temperature of 25-50° C. for 3 hours.

To a solution of the above amine hydrochloride (4.90 g, 14 mmol) inmethylene chloride (150 mL) is added (R)-2-bromo-3-methylbutanoic aciddiisopropyl amine salt (4.03 g, 14 mmol),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl, 2.70g, 14 mmol) and 1-hydroxy-7-azabenzotriazole (HOAT, 1.90 g, 14 mmol).The mixture is stirred at room temperature overnight and thenconcentrated in vacua. The residue is dissolved in ethyl acetate andwashed with water, dilute sodium bicarbonate, water, 1 N HCl, and thenbrine. The solution is dried over sodium sulfate and concentrated togive a solid. The solid is purified by flash chromatography (silica gel,2:1 hexane/ethyl acetate) to giveN-[2-(R)-2-bromo-3-methylbutanoylamino]-2-methylpropionyl]-O-benzyl-L-serineethyl ester of the formula

Alternately, the above condensation of (R)-2-bromo-3-methylbutanoic aciddiisopropylamine salt with the amine hydrochloride can be carried out inacetonitrile in the presence of CDMT (1.05 equiv.) andN-methyl-morpholine (1.5 equiv.) at a temperature of 5-25° C.

Similarly prepared are:

(b)N-[2-[(S)-2-acetylthio-3,3-dimethylbutanoylamino]-2-methylpropinyl]-O-benzyl-L-serineethyl ester

(c)N-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-O-benzyl-L-threonineethyl ester; m.p. 121-122° C.

(d)N-[2-[(S)-2-acetylthio-3-methoxybutanoylamino]-2-methylpropionyl]-O-benzyl-L-serineethyl ester; [α]_(D) ²⁰+14.9° (c=1.04, DMSO)

(e)N-[2-[(S)]-2-acetylthio-3-methylpentanoylamino]-2-methylpropionyl]-O-benzyl-L-serineethyl ester; [α]_(D) ²⁰−6.93° (c=1.09, CH₃OH)

(f)N-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-O-(3-trifluoromethylbenzyl)-L-serineethyl-ester

The starting O-(3-trifluoromethylbenzyl)-L-serine ethyl esterhydrochloride is prepared as follows:

To a suspension of sodium hydride (60% in oil, 3.04 g, 76 mmol) inN,N-dimethylformamide (60 mL) at 0° C. is added BOC-L-serine (7.80 g, 38mmol). The mixture is stirred for 1 hour and thenm-trifluoromethylbenzyl chloride (7.39 g, 38 mmol) is added. The mixtureis allowed to warm to room temperature and is stirred overnight. Themixture is quenched with water. Ethyl acetate is added and the mixtureis washed with brine, dried over MgSO₄ and concentrated to give a yellowoil which is purified by flash chromatography (SiO₂; hexane/ethylacetate) to give a clear oil. The residue is dissolved in ethanol (120mL), the solution is cooled to 0° C. and saturated with HCl gas for 5minutes. The mixture is allowed to warm to room temperature and stirredovernight. The mixture is concentrated to giveO-(4-trifluoromethylbenzyl)-L-serine ethyl ester hydrochloride.

(g)N-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-O-(4-fluorobenzyl)-L-serineethyl ester as an oil

(h)N-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-O-(4-fluorophenyl)-L-homoserineethyl ester as an oil

The starting O-(4-fluorophenyl)-L-homoserine ethyl ester hydrochlorideis prepared as follows:

To a solution of BOC-L-homoserine t-butyl ester (3.2 g, 11.6 mmol) intetrahydrofuran is added triphenylphosphine (7.59 g, 29 mmol),p-fluorophenol (2.08 g, 18.6 mmol) and1,1′-azobis(N,N-dimethylformamide) (3.2 g, 18.6 mmol). The mixture isstirred overnight, washed with brine, dried over MgSO₄, and the solventis removed to give an orange oil. The oil is purified by flashchromatography (SiO₂, 85% hexane/15% ethyl acetate) to give a clear oilwhich is dissolved in ethanol (100 mL) and the solution is saturatedwith HCl gas, then stirred overnight. The mixture is concentrated togive O-(4-fluorophenyl)-L-homoserine ethyl ester hydrochloride as awhite solid.

(i)N-[2-[(S)-2-acetylthio-3-methyl-butanoylamino]-2-methylpropionyl]-O-(3-fluorophenyl)-L-homoserineethyl ester

(j)N-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-O-benzyl-L-serinemorpholinocarbonylmethyl ester, purified by chromatography on silica gelwith hexane, ethyl acetate, methanol (20:70:10) as a white solid

The starting material is prepared as follows:

O-Benzyl-L-serine (10.0 g, 51.3 mmol), di-tert-butyl-dicarbonate (11.2g, 51.4 mmol) and 1 N sodium hydroxide (103 mL, 103 mmol) are stirredtogether in 100 mL of dioxane at room temperature for 16 hours. Themixture is concentrated in vacuo, taken up in water, acidified to pH 1with 6 N HCl and extracted with ethyl acetate. The organic layer iswashed with water, then brine, and dried over anhydrous magnesiumsulfate. The mixture is filtered and concentrated in vacuo to giveBOC-O-benzyl-L-serine as an oil. 4-(2-Chloroacetyl)morpholine (1.22 g,7.48 mmol) is added to a solution of BOC-O-benzyl-L-serine (2.20 g, 7.46mmol), triethylamine (0.75 g, 7.43 mmol) and sodium iodide (0.11 g, 0.73mmol) in 5 mL of N,N-dimethylformamide and the mixture stirred at roomtemperature for 2 hours. The mixture is diluted with ethyl acetate,washed with water, then with brine, and dried over anhydrous magnesiumsulfate. The mixture is filtered and concentrated in vacuo to give ayellow oil. The oil is chromatographed on silica gel with hexane:ethylacetate:methanol (35:60:5) to give BOC-O-benzyl-L-serinemorpholinocarbonylmethyl ester as a colorless oil. HCl gas is bubbledthrough a solution of the carbamate ester (1.72 g, 4.08 mmol) inmethylene chloride (50 mL) for 5 minutes and the mixture is stirred atroom temperature for 3 hours. The resulting mixture is concentrated invacuo to yield O-benzyl-L-serine morpholinocarbonylmethyl ester as afoam.

(k)N-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-O-benzyl-L-serinedimethylaminocarbonylmethyl ester, prepared and purified as describedfor compound of Example 1(j)

(l) N-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-O-benzyl-L-serinediethylaminocarbonylmethyl ester, prepared as described for compound ofExample 1(j) and purified by chromatography on silica gel with hexane,ethyl acetate, methanol (35:60:5)

(m)N-[2-[(S)-2-[(methoxyacetyl)thio]-3-methylbutanoylamino]-2-methylpropionyl]-O-benzyl-L-serineethyl ester; [α]_(D)−55.27°; (c=1.084, CH₃OH)

(n)N-[2-[(S)-2-[(morpholinoacetyl)thio]-3-methylbutanoylamino]-2-methylpropionyl]-O-benzyl-L-serineethyl ester; [a]_(D)−48.61° (c=1.098, CH₃OH)

(o)N-[2[(S)-2-acetylthio-2-(4-tetrahydropropanyl)acetylamino]2-methylpropionyl]-O-benzyl-L-serineethyl ester; [α]_(D) ²⁰−55.4° (c=0.83, DMSO)

The starting (D)-α-bromo-α-(4-tetrahydropyranyl)-acetic acid can beprepared as follows:

A solution of sodium nitrite (4.71 g, 68.3 mmol) in 35 mL of water isadded dropwise to a chilled (0° C.) solution of(D)-α-bromo-α-(4-tetrahydropyranyl)-glycine (J. Am. Chem. Soc., Vol.117, pp. 9375-9376 (1995) (7.05 g, 44.3 mmol) and 48% HBr (aq) (70 mL)in 35 mL of water. Upon completion of the addition, the mixture isallowed to warm to room temperature and stirred at room temperature for3 hours. The mixture is extracted with ethyl acetate; the organic layeris washed sequentially with water, 5% aqueous sodium thiosulfate, andbrine, then dried over anhydrous magnesium sulfate. The mixture isfiltered and concentrated in vacuo to yield(D)-α-bromo-α-(4-tetrahydropyranyl)-acetic acid as a solid.

(p)N-[2-[(S)-2-[(1-(1,2,4)-triazolyl)acetylthio]-3-methylbutanoylamino]-2-methylpropionyl]-O-benzyl-L-serineethyl ester; m.p. 106-107°; [α]_(D)−61.46° (c=1.09, CH₃OH)

(q)N-[2-[(S)-2-[(4-methylpiperazino)acetylthio]-3-methylbutanoylamino]-2-methylpropionyl]-O-benzyl-L-serineethyl ester; m.p. 95-96°; [α]_(D)−48.5° (c=0.935, CH₃OH)

(r)N-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-ethylbutanoyl]-O-benzyl-L-serineethyl ester; [α]_(D)−83.6° (c=1.07, CH₃OH).

(s)N-[2-[(S)-2-(morpholinoacetylthio)-3,3-dimethylbutanoylamino]-2-methylpropionyl]-O-benzyl-L-serineethyl ester; [α]_(D)−55.5° (c 1.008, DMSO)

(t)N-[2-[(S)-2-[(methoxyacetyl)thio]-3,3-dimethylbutanoylamino]-2-methylpropionyl]-O-benzyl-L-serineethyl ester; [α]_(D)−61.67° (c=1.024, DMSO)

(u)N-[2-[(S)-2-(acetylthio)pentanoylamino]-2-methylpropionyl]-O-benzyl-L-serineethyl ester

(v)N-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-O-(4-biphenylylmethyl)-L-serineethyl ester

EXAMPLE 2N-[2-[(S)-2-Acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-S-benzyl-L-cysteineethyl ester

The title compound is prepared similarly to Example 1 andre-crystallized from methyl t-butyl ether/hexane, m.p. 69.71° C.

The starting material is prepared as follows:

HCl (g) is bubbled into a solution of BOC-S-benzyl-L-cysteine (9.33 g,30 mmol) in ethanol (200 mL) for 15 minutes. The container is stopperedand stirred at room temperature overnight. The solvent is evaporated invacuo and the residue stirred in diethyl ether (150 mL) for 1.5 hours toyield S-benzyl-L-cysteine ethyl ester hydrochloride as a solid.

A mixture of S-benzyl-L-cysteine ethyl ester hydrochloride (7.98 g, 29mmol), BOC-α-methylalanine (5.89 g, 29 mmol), triethylamine (2.93 g, 29mmol), 1-hydroxybenzotriazole (HOBT, 3.92 g, 29 mmol) and EDCl (5.57 g,29 mmol) in methylene chloride (200 mL) is stirred under an argonatmosphere at room temperature overnight. The reaction mixture isevaporated to dryness and the residue is dissolved in ethyl acetate (200mL). The solution is washed with water (50 mL), 1 N HCl (50 mL), water(50 mL), 5% sodium bicarbonate (50 mL), water (50 mL) and finally brine(25 mL). The solution is then dried over sodium sulfate, filtered andevaporated to dryness to giveN-[2-(BOC-amino)-2-methylpropionyl]-S-benzyl-L-cysteine ethyl ester.

EXAMPLE 3N-[2-[(S)-2-Acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-(S)-2-amino-3-(benzylsulfonyl)-propionicacid ethyl ester

The above compound is prepared similarly to Example 1.

The starting material is prepared as follows:

To a solution of N-[2-(BOC-amino)-2-methylpropionyl]-S-benzyl-L-cysteineethyl ester (7.21 g, 17 mmol) in methylene chloride (250 mL) under anargon atmosphere is added m-chloro-perbenzoic acid (8.77 g, 51 mmol) andthe mixture is stirred overnight at room temperature. The mixture isevaporated to dryness and the residue is dissolved in ethyl acetate (300mL). The solution is washed with 5% sodium bicarbonate (3×50 mL), water(50 mL) and brine (25 mL). The solution is dried over sodium sulfate,filtered and evaporated in vacuo to giveN-[2-(BOC-amino)-2-methylpropionyl]-(S-)-2-amino-3-(benzylsulfonyl)-propionicacid ethyl ester.

EXAMPLE 4 (a)N-[2-[(S)-2-Acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-(S)-2-amino-3-(benzoylamino)-propionicacid methyl ester

A mixture of benzoyl chloride (0.085 mL, 0.73 mmol),N²-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-(S)-2,3-diaminopropionicacid methyl ester hydrochloride (0.29 g, 0.73 mmol) and triethylamine(0.15 mL, 1.49 mmol) in methylene chloride (10 mL) is stirred at roomtemperature for 16 hours. The reaction mixture is evaporated to drynessin vacuo, the residue is dissolved in ethyl acetate, and the solution iswashed with water, then with saturated sodium bicarbonate solution andbrine, dried over anhydrous magnesium sulfate, and evaporated to drynessto give an oil. The oil is chromatographed on silica gel with hexane,ethyl acetate (50:50) to yield the title compound as a white foam; m.p.48-54° C.

(b) Similarly prepared isN²-[2[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-(S)-2-amino-3-(benzenesulfonamido)propionicacid methyl ester; m.p. 47-51° C.; [α]_(D) ²⁰−41.72 (c=1.03, CH₃OH).

The starting material is prepared as follows:

A mixture of (S)-2-amino-3-(BbC-amino)-propionic acid methyl esterhydrochloride (4.6 g, 2.1 mmol), N-CBZ-a-methylalanine (5.0 g, 2.1mmol), HOAT (2.87 g, 2.1 mmol), EDCl (4.02 g, 2.1 mmol) andtriethylamine (2.93 g, 2.1 mmol) in methylene chloride (50 mL) isstirred at room temperature for 16 hours. The reaction mixture is washedwith brine, dried over anhydrous magnesium sulfate and concentrated invacuo. The resulting oil is chromatographed on silica gel with hexaneand ethylacetate (1:1) to yieldN²-[2-(CBZ-amino)-2-methylpropionyl]-(S)-2-amino-3-(BOC-amino)-propionicacid methyl ester as a white foam; m.p. 100-101° C.

A mixture of the above product (2.14 g, 4.90 mmol) and 10% palladium oncharcoal (0.27 g) in ethanol (50 mL) is hydrogenated under 45 psipressure in a Parr bottle for 4 hours. The mixture is filtered through apad of Celite and concentrated in vacuo to giveN²-[2-amino-2-methylpropionyl]-(S)-2-amino-3-(BOC-amino)-propionic acidmethyl ester hydrochloride as an oil.

A solution of the above product (2.28 g, 8.09 mmol),(R)-2-bromo-3-methylbutanoic acid diisopropyl amine salt (2.16 g, 7.13mmol), EDCl (1.43 g, 7.49 mmol) and HOAT (1.15 g, 8.52 mmol) inmethylene chloride (75 mL) is stirred at room temperature for 16 hours.The reaction mixture is evaporated to dryness in vacuo and the residuetaken up in ethyl acetate. The ethyl acetate solution is washed withwater, saturated sodium bicarbonate solution and brine, and then driedover anhydrous magnesium sulfate, and concentrated in vacuo. Theresulting oil is chromatographed on silica gel with hexane and ethylacetate (40:60) to giveN²-[2-[(R)-2-bromo-3-methylbutanoylamino]-2-methylpropionyl]-(S)-2-amino-3-(BOC-amino)-propionicacid methyl ester as a white foam.

A mixture of the above product (1.31 g, 2.82 mmol) and potassiumthioacetate (1.28 g, 11.2 mmol) in tetrahydrofuran (50 mL) is stirred atroom temperature for 4 hours and diluted with ethyl acetate. The mixtureis washed with water, saturated sodium bicarbonate solution, brine andthen dried over magnesium sulfate. The reaction mixture is concentratedto dryness in vacuo and the resulting oil is chromatographed on silicagel with hexane and ethyl acetate (40:60) to giveN-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-(S)-2-amino-3-(BOC-amino)-propionicacid methyl ester.

Hydrogen chloride gas is bubbled through a solution of the abovecompound (1.01 g, 2.19 mmol) in 50 mL of methylene chloride for about 5minutes, the mixture is stirred at room temperature for 3 hours, andthen concentrated in vacub to yieldN²-[2-[(S)-2-acetylthio-3-methylbutanoylamino]-2-methylpropionyl]-(S)-2,3-diaminopropionicacid methyl ester hydrochloride.

EXAMPLE 5

To a solution of1-[(R)-2-bromo-3-methylbutanoylamino]cyclopentanecarboxylic acid (1 g,3.42 mmol), O-benzyl-L-serine ethyl ester hydrochloride (0.89 g, 3.42mmol), dicyclohexylcarbodiimide (0.7 g, 3.42 mmol) and1-hydroxy-7-azabenzotriazole (0.47 g, 3.42 mmol) in methylene chlorideis added triethylamine (0.48 mL, 3.42 mmol). The mixture is stirred for24 hours and then washed with brine and concentrated in vacuo to give alight yellow oil. The residue is purified by flash chromatography(silica gel hexane/ethyl acetate) to giveN-[1-(R)-2-bromo-3-methylbutanoylamino]-cyclopentanecarbonyl]-O-benzyl-L-serineethyl ester of the formula

The bromo compound (0.7 g, 1.41 mmol) is dissolved in tetrahydrofuran(50 mL) and potassium thioacetate (0.19 g, 1.69 mmol) is added. Themixture is stirred at room temperature for 18 hours and then dilutedwith ethyl acetate and washed with brine, dried over magnesium sulfateand concentrated in vacuo to give yellow oil. The crude material ispurified by flash chromatography (silica gel, hexane/ethyl acetate) togive a semi-solid which is triturated with hexane to yieldN-[1-[(S)-2-acetylthio-3-methylbutanoylamino]-cyclopentanecarbonyl]-O-benzyl-L-serineethyl ester of the formula

The 1-[(R)-2-bromo-3-methylbutanoylamino]cyclopentanecarboxylic acidstarting material is prepared essentially by methodology described in WO99/55726 by condensation of (R)-2-bromo-3-methylbutanoic aciddiisopropylamine salt (prepared from L-valine) with cycloleucine methylester hydrochloride.

EXAMPLE 6 (a)N-[2-[(S)-2-Mercapto-3-methylbutanoylamino]-2-methylpropionyl]-O-benzyl-L-serine

To a solution of the S-acetyl ethyl ester of Example 1 (0.47 g, 1 mmol)in methanol (10 mL) is added 1 N sodium hydroxide (5.0 mL, 5 mmol). Themixture is stirred at room temperature for 4 hours, acidified to pH 1with 1 N HCl and then concentrated in vacuo. To the residue is addedethyl acetate. The mixture is washed with 1 N NaOH. The combined aqueousphase is then acidified and extracted with ethyl acetate. The organicphase is washed with brine, dried over sodium sulfate and thenconcentrated in vacuo. Trituration with hexane yields a white foam; m.p.57-70° C.; [α]_(D) ²⁰−16.8° (c=1.032, DMSO); MS (M+H): 397.

Similarly prepared are the following:

(b)N-[1-[(S)-2-mercapto-3-methylbutanoylamino]-cyclopentanecarbonyl]-O-benzyl-L-serine;m.p. 132-136° C. (crystallized from hexane/t-butylmethyl ether)

(c)N-[2-[(S)-2-mercapto-3-methylbutanoylamino]-2-methylpropionyl]-S-benzyl-L-cysteine;m.p. 81-87° C.; [α]_(D) ²⁰−37.87 (c=0.545, DMSO)

(d)N-[2-[(S)-2-mercapto-3-methylbutanoylamino]-2-methylpropionyl]-O-benzyl-L-threonine;m.p. 61-64° C.

(e)N-[2-[(S)-2-mercapto-3,3-dimethylbutanoylamino]-2-methylpropionyl]-O-benzyl-L-serine;m.p. 128-130° C.; [α]_(D)−2.46 (c=1.06, DMSO)

(f)N-[2-[(S)-2-mercapto-3-methibutanoylamino]-2-methylpropionyl]-O-(4-fluorobenzyl)-L-serine;m.p. 50-54° C.

(g)N-[2-[(S)-2-mercapto-3-methylbutanoylamino]-2-methylpropionyl]-O-(4-fluorophenyl)-L-homoserine;m.p. 127-128° C.

(h)N-[2-[(S)-2-mercapto-3-methylbutanoylamino]-2-methylpropionyl]-O-(3-fluorophenyl)-L-homoserine;m.p. 5°-56° C.

(i)N-[2-[(S)-2-mercapto-3-methoxybutanoylamino]-2-methylpropionyl]-O-benzyl-L-serine;[α]_(D) ²⁰+18.85 (c=0.997, DMSO)

(j)N-[2-[(S)-2-mercapto-2-(4-tetrahydropyranyl)acetylamino]-2-methylpropionyl]-O-benzyl-L-serine;m.p. 184-189° C.; [α]_(D) ²⁰−24.94 (c=1.013, DMSO)

EXAMPLE 7 (a)N-[2-[(S)-2-mercapto-3-methylbutanoylamino]-2-methylpropionyl]-S-benzyl-L-cysteineethyl ester

Under an argon atmosphere, the thioacetyl compound of Example 2 (0.48 g,1.0 mmol) is dissolved in absolute EtOH (5 mL) and treated with 1 N NaOHof (1.0 mL, 1.0 mmol). The mixture is stirred for 4 hours at roomtemperature before treatment with 1 N HCl until pH 3. The mixture isevaporated to remove most of the EtOH and the aqueous residue isextracted with EtOAc (2×10 mL). The combined extracts are washed withH₂O (5 mL) and then with brine solution (5 mL). The solution is driedover Na₂SO₄, filtered and concentrated in vacuo. The product solidifiesfrom tert-butyl methyl ether/hexane to give product; m.p. 87-91° C.

(b) Similarly prepared isN-[2-[(S)-2-mercapto-2-(4-tetrahydropyranyl)acetylamino]-2-methylpropionyl]-O-benzyl-L-serineethyl ester; m.p. 85-93° C.; [α]_(D)−37:21° (c=1.012, DMSO)

(c) Similarly Prepared isN-[2-[(S)-2-mercapto-3,3-dimethylbutanoylamino]-2-methylpropionyl]-O-benzyl-1-serineethyl ester; oil; [α]_(D)−20.90 (c=1.025, DMSO)

(d) Similarly Prepared isN-[2-[(S)-2-mercapto-3-methylbutanoylamino]-2-ethylbutanoyl]-O-benzyl-1-serineethyl ester; [α]_(D)−31.48° (c=0.955, CH₃OH)

1. A pharmaceutical composition comprising (a) a compound of the formula(I)

wherein R represents hydrogen, lower alkyl, carbocyclic or heterocyclicaryl-lower alkyl or cycloalkyl-lower alkyl; R₁ represents lower alkyl,cycloalkyl, carbocyclic or heterocyclic aryl, or biaryl; or R₁represents (cycloalkyl, carbocyclic aryl, heterocyclic aryl orbiaryl)-lower alkyl; alk represents lower alkylene; R₃ representshydrogen or acyl; R₄ represents hydrogen, optionally substituted loweralkyl, carbocyclic or heterocyclic aryl, (carbocyclic or heterocyclicaryl)-lower alkyl, cycloalkyl, cycloalkyl-lower alkyl, biaryl,biaryl-lower alkyl; oxacycloalkyl, thiacycloalkyl, azacycloalkyl, or(oxacycloalkyl, thiacycloalkyl or azacycloalkyl)-lower alkyl; R₅represents hydrogen or lower alkyl; or R₄ and R₅ together with thecarbon atom to which they are attached, represent cycloalkylidene,benzo-fused cycloalkylidene; or 5- or 6-membered (oxacycloalkylidene,thiacycloalkylidene or azacycloalkylidene), each optionally substitutedby lower alkyl or aryl-lower alkyl; R₆ represents lower alkyl,carbocyclic or heterocyclic aryl, (carbocyclic or heterocyclicaryl)-lower alkyl, cycloalkyl, cycloalkyl-lower alkyl, biaryl orbiaryl-lower alkyl; R₇ represents lower alkyl, (carbocyclic orheterocyclic aryl)-lower alkyl, cycloalkyl-lower alkyl or biaryl-loweralkyl; or R₆ and R₇, together with the carbon atom to which they areattached, represent 3- to 10-membered cycloalkylidene which may besubstituted by lower alkyl or aryl-lower alkyl or may be fused to asaturated or unsaturated carbocyclic 5- to 7-membered ring; or 5- or6-membered (oxacycloalkylidene, thiacycloalkylidene orazacycloalkylidene), each optionally substituted by lower alkyl oraryl-lower alkyl; or 2,2-norbonylidene; X represents —O—, —S(O)_(r)—,—NHSO₂—, or —NHCO—; n is zero, one or two; and COOR₂ represents carboxylor carboxyl derivatized in form of a pharmaceutically acceptable ester;or a disulfide derivative derived from a said compound wherein R₃ ishydrogen; or a pharmaceutically acceptable salt thereof; and (b) atleast one second therapeutic agent selected from angiotensin II receptorantagonists, renin inhibitors, calcium channel blockers, aldosteronesynthase inhibitors, diuretics, vasopressin receptor antagonists,cardiotonic drugs, endothelin antagonists and ECE inhibitors,anti-atherosclerotic agents, cholesterol absorption inhibitors, statinHMG CoA reductase inhibitors, nicotinic acid derivatives, thyromimeticagents, and antidiabetic agents.
 2. The composition of claim 1, whereinthe compound has the formula (Ia):


3. The composition of claim 1, wherein the compound has the structure:


4. The composition of claim 1, wherein the angiotensin II receptorantagonists are selected from valsartan, losartan, candesartan,eprosartan, irbesartan and telmisartan.
 5. The composition of claim 1,wherein the blockers are selected from bisoprolol, propanolol, atenolol,sotalol and metoprolol.
 6. The composition of claim 1, wherein thecalcium channel blockers are selected from amlodipine, verapamil,diltiazem, bepridil, felodipine, isradipine, nicardipine, nifedipine,nimodipine and nisoldipine.
 7. The composition of claim 1, wherein thealdosterone synthase inhibitors are selected from eplerenone,(+)-fadrozole (WO 01/76574), spironolactone and canrenone.
 8. Thecomposition of claim 1, wherein the diuretics are selected fromfurosemide, hydrochlorothiazide, indapamide, metazolone, amiloride andtriamterene
 9. The composition of claim 1, wherein the vasopressinreceptor antagonists are selected from OPC 21268, SR 49059, SR121463A,SR49059, VPA985, OPC31260 and YM087.
 10. The composition of claim 1,wherein the statin HMG CoA reductase inhibitors are selected fromatorvastatin, fluvastatin, lovastatin, pravastatin, simvastatin andpitavastatin.
 11. The composition of claim 1, wherein the antidiabeticagents are selected from repaglinide, nateglinide, metformin,rosiglitazone, pioglitazone, glyburide, glipizide, glimepiride, DPP728,LAF237, NH622 and DRF4158.
 12. A method of treating or inhibitingcardiovascular disorders in mammals comprising administering to a mammalin need thereof an effective amount of the pharmaceutical composition ofclaim
 1. 13. The method according to claim 12 for the treatment ofhypertension, edema, salt retention or congestive heart failure.